Application Work AW IC ES6-0009-112019 Determination of free and total sulfite in food and beverages by IC with DC mode electrochemical detection operative for laboratories where sulfite analysis is required in Branch a wide range of food and beverages products. Food, stimulants, beverages, flavors This AW describes an innovative, fast and accurate IC Keywords method based on DC mode electrochemical detection. It IC, DC, electrochemical detection, amperometric detection, works with a Metrohm unique working electrode conditioning 930 889, sold, drinks, soft drinks, food, alcohols, sulfite, function, which has been implemented for first time in MagIC beverages, Metrosep Carb 2-150/4.0, branch 7 Net 3.3 version. The analytical procedure consists of an alkaline extraction Summary followed by anion exchange separation using Metrosep Carb Sulfite is a widely used preservative added to prevent 2 - 150/4,0 column and NaOH-NaOAc (300mM-300mM) browning or oxidation to a wide range of food and beverages eluent, followed by DC mode electrochemical detection. For products as meat, fish, legumes, soft drinks, wine or beer. sulfite preservation during long determination series, the Many individuals are sensitive to sulfite additives and may system is automated with 889 IC Sampler Center with cooling experience a range of allergic reactions. For certain people, function. sulfite has also been reported as the cause of some asthmatic A great diversity of food and beverages samples was responses. For this reason, both FDA and EU laws require analyzed with successful sulfite recovery values in all cases that the presence of sulfites be declared on food labels when under the conditions of a single chromatographic method. In the sulfite concentration is higher than 10 mg/L. appendix of this AW, some chromatograms are shown, The optimized Monier–Williams (OMW) Official Method revealing unique selectivity values and a performance of 990.28, has been traditionally used for sulfites quantification sulfite stability with optimal results for the analytical routine. in most matrixes, but this method is time-consuming, labor-intensive and has a method detection limit at the regulatory Samples labeling threshold. Several foodstuff matrices, eg. chickpea, mustard, cherries, More efficient and accurate methods have been tested using capers, canned garlic, chilli pepper, red wine. stability solution extraction to overcome sulfite chemical Instruments instability in aqueous solution followed by different detection techniques. Methods based on ion chromatography with conductivity detection show lack of selectivity and long analysis time due to the need to separate sulfite from sulfate as well as from many other coeluting organic interferents. Some other chromatographic methods have been tested based on ion-exclusion chromatography with DC mode electrochemical detection (AOAC Official Method 990.31) resulting in recognized errors due to gradual decrease in detector sensitivity during multiple injections. Even ion exclusion chromatography with PAD based methods show the need to polish working electrode quite often due to background rinsing and baseline noise. Automated discrete analysis methods have also been reported for sulfite analysis. The limitation of these methods is their strong dependence on the kind of matrix to be analyzed. This is the reason because these methods are not Application Work AW IC ES6-0009-112019 Determination of free and total sulfite in food and beverages by IC with DC mode electrochemical detection 930 Compact IC Flex Oven/Deg 2.930.2160 Sample preparation 889 IC Sample Center – Cool 2.889.0020 Fresh samples prepared with fresh working stabilization IC Amperometric Detector 2.850.9110 solution IC equipment Wall-Jet cell: without 6.1257.000 Weigh 1 g of crushed sample in a Falcon tube. Add the electrodes necessary volume of the working stabilization solution until it weighs 30 g (sample dilution 1/30). Homogenize 1 min at Ag/AgCl reference electrode for 6.1257.720 25000 rpm with ultra-turrax homogenizer. Finally, centrifuge 6.1257.xxx sample at 4000 rpm Au working electrode, 3 mm 6.1257.210 Metrosep Carb 2 150/4.0 6.1090.420 Analysis Metrosep Carb 2 Guard 6.1090.500 Every solution was filtrated with 0.2 µm before filling 2 mL vial MagIC Net™ 3.3 Professional samples with septum. It is important to fill completely the 6.6059.332 sample vial in order to eliminate head-space air. Reagents • Sodium hydroxide solution, 49 – 51% in water, eluent Eluent Flow 0.5 mL/min for IC. CAS 1310-73-2. Supelco: 72064 Detector Mode DC • Sodium Acetate anhydrous ≥ 99%. CAS 127-09-3. E(V) 300 mV Sigma-Aldrich: S2889 Range AUTO • Formaldehyde solution ≥34.5 wt.%. CAS 50-00-0. Damping OFF Sigma-Aldrich 15512 Detector temperature 35 ºC • Sodium sulfite, BioUltra anhydrous ≥ 98% (RT). CAS Col. Oven temperature 35 °C 7757-83-7. Sigma-Aldrich: 71988 Channel measurement • Current (nA) Soda lime p.a., with indicator, pellets; CAS 8006-28-8; Sample Loop (Partial Loop Mode) 3 µL Sigma-Aldrich: 72073 • Sample cooling temperature 6 ºC Ultrapure water (UPW) resistivity >18.2 MΩ·cm (25 °C) type I grade (ASTM D1193) from Flex 5 ELGA water Nº cycles CV treatment 6 system Start potential - 0.6 V Solutions End potential 0.7 V Sweep rate 0.1 V/s Eluent Sodium Hydroxide 300.0 mmol/L Sodium Acetate 300.0 mmol/L CV mode Full cycle CO2 free ultrapure water Range 2 mA Rinsing Solution UPW Stock stabilization Formaldehyde 1.00 mol/L Calculation solution Sodium Hydroxide 0.20 mol/L Automatic integration using peak areas evaluated with MagIC Working stabilization Formaldehyde 1.00 mmol/L NetTM 3.3 solution (dil: 1/1000 Sodium Hydroxide 0.20 mmol/L Results of stock solution) See appendix with chromatograms below Stabilization solutions Comments Always prepare daily fresh solutions. This fast and accurate IC method overcomes all the Prepare a stabilization stock solution from commercial limitations found in the sulfite analysis based on the different reagents and then dilute 1:1000, with well degassed UPW, to analytical methodologies used to date and is valid for any obtain working stabilization solution. kind of food and beverages. Standard solutions It is necessary to work with MagIC Net 3.3 version to get all the potential and stability in long terms of analytical routine, Sulfite anion solution from 0.2 to 7.5 mg/L with working due to the new command CV treatment, which allows surface stabilization solution working electrode cleaning. Sample vials must be completely filled with prepared samples and closed immediately to prevent sulfite oxidation. Page 2 of 8 Application Work AW IC ES6-0009-112019 Determination of free and total sulfite in food and beverages by IC with DC mode electrochemical detection The use of 889 IC Sample Center - Cool permits a long stability of prepared samples. The sample cooling temperature at 6 ºC showed as an optimal value for sample stability for several hours. It is important to remark that the combination of a very high capacity column, as Metrosep Carb 2, and the eluent NaOH-NaOAc (300mM-300mM) revealed an important improvement of peak resolution between Formaldehyde and Sulfite, reducing sample matrix effect as well as a very precise repeatability of retention times. Stabilization solutions should be freshly prepared to get the optimal stability of sulfite in samples as well as in sulfite standards. Eluent and stabilization solutions must be prepared from well degassed UPW just before launching the analytical sequence. References [1] AW IC ES6-0007-082017_G Sulfite determination in Cider by amperometric detection with Metrosep Carb 2. [2] AW IC CH6-1089-032012 Determination of sulfite using the Amperometric Detector with the DC Mode and two different stabilization solutions. [3] AN-S-225 Sulfite in the presence of standard anions in beer on the column Metrosep A Supp 10 - 100/4.0. [4] AW IC ES6-0003-092013_G Determination of Lactose in Lactose-free Yogurt, with Pulsed Amperometric Detection Ion Chromatography (PAD-IC). Date 22th October 2019 Authors IC Department Metrohm Hispania Page 3 of 8 Application Work AW IC ES6-0009-112019 Determination of free and total sulfite in food and beverages by IC with DC mode electrochemical detection Appendix Example calibration chromatograms for 3 µL injection volume Fig 1 . Injection of 3 µL sulfite standard 7.5 mg/L Fig 2 . Injection of 3 µL sulfite standard 0.2 mg/L Page 4 of 8 Application Work AW IC ES6-0009-112019 Determination of free and total sulfite in food and beverages by IC with DC mode electrochemical detection Fig 3. Overlay of sulfite standards chromatograms Fig 4. Calibration curve for sulfite standards from 0.2 to 7.5 mg/L Page 5 of 8 Application Work AW IC ES6-0009-112019 Determination of free and total sulfite in food and beverages by IC with DC mode electrochemical detection Sample Chromatograms for 3 µL injection volume: Canned garlic Fig 5. Canned garlic sample dil: 1/30 Fig 6. Fortified canned garlic sample dil:1/30 Fig 7. Overlay of canned garlic and fortified canned garlic Page 6 of 8 Application Work AW IC ES6-0009-112019 Determination of free and total sulfite in food and beverages by IC with DC mode electrochemical detection Sample Chromatograms for 3 µL injection volume: Mustard Fig 8. Mustard sample dil: 1/30 Fig 9. Fortified mustard sample dil: 1/30 Fig 10. Overlay of mustard and fortified mustard sample dil: 1/30 Page 7 of 8 Application Work AW IC ES6-0009-112019 Determination of free and total sulfite in food and beverages by IC with DC mode electrochemical detection Sample chromatograms for 3 µL injection volume: Red wine Fig 11. Red wine sample dil: 1/30 Fig 12. Fortified red wine sample (dil: 1/30) Fig 13. Overlay of red wine and fortified red wine sample (dil: 1/30) Page 8 of 8